This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The phosphoinositide kinase related protein kinases (PIKK) mTOR, ATM and ATR have crucial cellular functions. mTOR is a central effector kinase in the PI3K/Akt signaling pathway that is frequently dysregulated in human cancers. ATM and ATR are essential cell cycle checkpoint kinases in the DNA damage signaling pathway, protecting cells against DNA damage and oxidative stress. Inhibitors against mTOR are currently in clinical cancer therapy trials. Additional selective inhibitors against these enzymes could have numerous applications in laboratory experiments and might be of clinical value for the treatment of cancer. mTOR, ATM and ATR are large proteins with molecular weights of 288, 350, and 300kDa, respectively. They contain a conserved C-terminal region, consisting of a FAT domain, a catalytic PIKK protein kinase domain and a FATC domain. The catalytic domain of all three proteins shares sequence homology with phosphoinositide kinases (PIK), such as phosphatidylinositol 3- and 4kinases (PI3K, PI4K). All PIK and PIKK catalytic domains are inhibited by the small-molecule inhibitor wortmannin, which binds to the ATP binding site. Despite the functional importance of PIKKs, only one protein structure of class I PI3K is currently available as a template for this enzyme family. Here, we propose to determine X-ray crystal structures of the catalytic domains of PIKs and PIKKs to investigate the structural basis of enzymatic catalysis and substrate specificity. We will test the hypothesis that all family members contain a structurally related ATP binding site that binds wortmannin in a similar position, and that the substrate-binding site of PI3K and PI4K is structurally related to each other, whereas the substrate-binding site of the PIKKs is unrelated.